Chromosome biology lecture 6

Question 1

Site specific recombination (SSR) overview

Answer 1

• Protein binds recognition site + catalyses exchange w/ another site that is recognised by the same protein
• Basis of recognition = protein DNA + protein-protein interaction

Question 2

SSR

Answer 2

• Used by bacteriophage lamda
• Minimum requirement = small core DNA site (inverted repeat of recombinase bs)
• Monomer of recombinase binds each of the repeats

Question 3

Outcomes of SSR

Answer 3

• Depends on relative orientation of 2 core sites

- Excision, integration or inversion

Question 4

Roles in nature

Answer 4

1. Flp (maintaining copy no. of 2 micron plasmid in yeast, ssrecombinase encoded by 2 micron plasmid, recognises FRT, Flp ↑ copy no by recombining 2 FRT sites on plasmid, single origin of replication, recombine 1 newly replicated FRT, resolved)
2. Resolving chromosome dimers (circular chromosomes = hard to segregate chromosomes to daughter cell, bifunctional fork move away from OriC, resolution → crossover)
3. Integration + excision of bacteriophage (life cycle, integration + excision of DNA depends on lambda integration)
4. ss inversion system (on/off switch for gene expression, e.g. salmonella, Hin drives switch btw 2 different flagella filament proteins FljB/C)
5. Resolution of transposition intermediates (Tn3 transposon)
6. Acquisition of new drug resistance (e.g. integrons, incorporate exogenous ORF by SSR + convert to functional genes, intl + attl, array of gene

Question 5

SS recombinase families

Answer 5

1. Tyr recombinase family
2. Ser “ “
   - Common = (inverted repeat bound by recombinase, 1 phosphodiesterase bond cleaved, SN2 reaction)
   - Difference = (Tyr catalyses 2 pairs of strand cleavage, Ser = both simultaneously)

Question 6

Tyr recombinase family

Answer 6

• Flp, Cre v similar = overall 2-fold symmetry
• Clamp around DNA substrate
• CTD a-helical + has AS residues, NTD ↑ varied
• Catalytic domain = 6 conserved residues
• 1 strand cleaved in each ds DNA molecule
• Each cleavage → phosphoTyr linkage btw recombinase 3’ end of DNA
• Form HJ intermediate, resolved
• Synaptic complex

Question 7

Ser recombinase family

Answer 7

• Conserved domain, 4 motifs w/ catalytic residues in A + C
• NTD = AS, CTD = DBD
• Asp + 2Arg crucial for catalysis
• 180o rotation of 2 subunits within complex

Question 8

Controlling direction of reaction

Answer 8

• DNA must be bent so 2 monomers interact
• Tyr recombinase, synaptic complex has spacer antiparallel → predictive strand exchange
• Ser recomb also needs bp of comp 2 bp to properly align 3’OH w/ 5’ phosphoser
• Sites = parallel, unproductive synaptic complex → all 4 strands cleavage + 180o rotation

Question 9

Controlling direction of recombination

Answer 9

• Sites in direct repeat in same DNA combine → deletion
• Diffrent sites recombine → integration
• Many recomb systems flip balance using accessory proteins + DNA seq elements
  E.g. = bacteriophage lamda from E coli

Question 10

Filter to sense connectivity btw distant DNA sites

Answer 10

• Resolvases like Tn3 avoid catalysing inversion + intermolecular recom by only being active in a specific complex
• Different no. of DNA crossings needed